1. Field of the Invention
The present invention concerns biologically active compounds related to the bryostatin family of compounds, and to methods of preparing and utilizing the same.
2. Introduction
Cancer is a major cause of death in the developed countries, with more than 500,000 human fatalities occurring annually in the United States. Cancers are generally the result of the transformation of normal cells into modified cells that proliferate excessively, leading to the formation of abnormal tissues or cell populations. In many cancers, cell proliferation is accompanied by dissemination (metastasis) of malignant cells to other parts of the body which spawn new cancerous growths. Cancers can significantly impair normal physiological processes, ultimately leading to patient mortality. Cancers have been observed for many different tissue and cell types, with cancers of the lung, breast, and colorectal system accounting for about half of all cases.
Currently, about one-third of cancer patients can be cured by surgical or radiation techniques. However, these approaches are most effective with cancerous lesions that have not yet metastasized to other regions of the body. Chemotherapeutic techniques currently cure another 17% of cancer patients. Combined chemotherapeutic and non-chemotherapeutic protocols can further enhance prospects for full recovery. Even for incurable cancer conditions, therapeutic treatments can be useful to achieve remission or at least extend patient longevity.
Numerous anticancer compounds have been developed over the past several decades (e.g., Katzung, 1998; Wilson et al., 1991; Hardman et al., 1996). While these compounds comprise many different classes that act by a variety of mechanisms, one general approach has been to block the proliferation of cancerous cells by interfering with cell division. For example, anthracyclines, such as doxorubicin and daunorubicin, have been found to intercalate DNA, blocking DNA and RNA synthesis and causing strand scission by interacting with topoisomerase II. The taxanes, such as Taxol(trademark) and Taxotere(trademark), disrupt mitosis by promoting tubulin polymerization in microtubule assembly. Cis-platin forms interstrand crosslinks in DNA and is effective to kill cells in all stages of the cell cycle. As another example, cyclophosphamide and related alkylating agents contain di-(2-chloroethyl)-amino groups that bind covalently to cellular components such as DNA.
The bryostatins (Formula A) are a family of naturally occurring macrocyclic compounds originally isolated from marine bryozoa. Currently, there are about 20 known natural bryostatins which share three six-membered rings designated A, B and C, and which differ mainly in the nature of their substituents at C7 (ORA) and C20 (RB) (Pettit, 1996). 
The bryostatins exhibit potent activity against a broad range of human cancer cell lines and provide significant in vivo life extensions in murine xenograft tumor models (Pettit et al., 1982; Hornung et al., 1992; Schuchter et al., 1991; Mohammad et al., 1998). Doses that are effective in vivo are extremely low, with activities demonstrated for concentrations as low as 1 xcexcg/kg (Schuchter et al., 1991). Among additional therapeutic responses, the bryostatins have been found to promote the normal growth of bone marrow progenitor cells (Scheid, 1994; Kraft, 1996), provide cellular protection against normally lethal doses of ionizing radiation (Szallasi, 1996), and stimulate immune system responses that result in the production of T cells, tumor necrosis factors, interleukins and interferons (Kraft, 1996; Lind, 1993). Bryostatins are also effective in inducing transformation of chronic lymphocytic leukemia cells to a hairy cell type (Alkatib, 1993), increasing the expression of p53 while decreasing the expression of bcl-2 in inducing apoptosis in cancer cells (Maki, 1995; Mohammad, 1995) or at least pre-disposing a cell towards apoptosis, and reversing multidrug resistance (MDR) (Spitaler, 1998).
At the molecular level, bryostatins have been shown to competitively inhibit the binding of plant-derived phorbol esters and endogenous diacyl glycerols to protein kinase C (PKC) at nanomolar to picomolar drug concentrations (DeVries, 1998), and to stimulate comparable kinase activity (Kraft, 1986; Berkow, 1985; Ramsdell, 1986). Unlike the phorbol esters, however, the bryostatins do not act as tumor promoters. Thus, the bryostatins appear to operate through a mode of action different from, and complementary to, the modes of action of established anticancer agents; human clinical trials are presently evaluating bryostatin combination therapy with cisplatin or taxol.
Various studies have demonstrated good affinity for bryostatins in which RA is hydroxyl, acetyl, pivaloyl, or n-butanoate, and RB is H, acetyl, n-butanoate, or 2,4-unsaturated octanoate, as measured by PKC binding assay (Wender et al., 1988). The double bond between C13 and C30 can be hydrogenated or epoxidized without significant loss of binding affinity. Hydrogenation of the C21-C34 alkene or acetylation of the C26 hydroxyl, on the other hand, can significantly reduce binding affinity. Inversion of the stereoconfiguration at C26 leads to modest loss of activity (approx. 30-fold) and the suggestion that the methyl group may limit rotation of bonds proximate to the methyl group and contribute to the apparent high binding affinity observed for the bryostatins. Elimination of the hydroxyl at C19 (with concomitant omission of the C20 RB group) causes an approximately 100-fold to 200-fold decrease in binding. Likewise, impairing the accessibility of the C26 hydroxymethyl moiety by replacement of the C26 hydroxyl, or by replacing the methyl or hydrogen substituents of C26 with a tert-butyl or similar bulky substituent, has been proposed for diminishing toxicity (Blumberg et al., 1997).
Although the bryostatins have been known for some time, their low natural abundance, difficulties in isolation and severely limited availability through total synthesis have impeded efforts to elucidate their mode of action and to advance their clinical development. Recently, synthetic analogues of bryostatin were reported wherein the C4-C14 spacer domain was replaced with simplified spacer segments using a highly efficient esterification-macrotransacetalization (Wender et al., 1998a, 1998b). The reported analogues retained orientation of the C1-, C19-, C26-oxygen recognition domain as determined by NMR spectroscopic comparison with bryostatin and varying degrees of PKC-binding affinity. The one analogue tested for in vitro inhibition in human tumor cell lines was reported to posess significant activity. It has remained, however, desired to provide new, simplified, and more readily accessible synthetic agents based on the bryostatin structure to further elucidate the molecular basis of bryostatin""s activity and develop improved and more readily available clinical candidates, especially for anticancer applications.
One aspect of the present invention concerns simplified bryostatin analogues, i.e., the compounds represented by Formula I: 
wherein:
R20 is H, OH, or O2CRxe2x80x2;
R21 is xe2x95x90CRaRb or R21 represents independent moieties Rc and Rd where:
Ra and Rb are independently H, CO2Rxe2x80x2, CONRcRd or Rxe2x80x2;
Rc and Rd are independently H, alkyl, alkenyl or alkynyl, or (CH2)nCO2Rxe2x80x2 where n is 1, 2 or 3;
R26 is H, OH or Rxe2x80x2;
each Rxe2x80x2 being independently selected from the group: alkyl, alkenyl or alkynyl, or aryl, heteroaryl, aralkyl or heteroaralkyl;
L is a straight or branched linear, cyclic or polycyclic moiety, containing a continuous chain of preferably from 6 to 14 chain atoms, which substantially maintains the relative distance between the C1 and C17 atoms and the directionality of the C1C2 and C16C17 bonds of naturally-occurring bryostatin; and
Z is xe2x80x94Oxe2x80x94 or xe2x80x94N(H)xe2x80x94;
and the pharmaceutically acceptable salt thereof.
In a preferred aspect of the recognition domain in this embodiment R26 is H or methyl, particularly when R21 is xe2x95x90C(H)CO2Rxe2x80x2. Especially preferred are the compounds where R26 is H. A preferred upper limit on carbon atoms in any of Rd, Re and Rxe2x80x2 is about 20, more preferably about 10 (except as otherwise specifically noted, for example, with reference to the embodiment of the invention where a preferred R20 substituent has about 9 to 20 carbon atoms). In a preferred aspect of the spacer domain of this embodiment, L contains a terminal carbon atom that, together with the carbon atom corresponding to C17 in the native bryostatin structure, forms a trans olefin.
Another aspect of the invention concerns the simplified bryostatin analogues represented by Formulae II-V: 
wherein:
R3 is H, OH or a protecting group;
R6 is H, H or xe2x95x90O;
R8 is selected from the group: H, OH, Rxe2x80x2, xe2x80x94(CH2)nO(O)CRxe2x80x2 or (CH2)nCO2-haloalkyl where
n is 0, 1, 2, 3, 4 or 5;
R9 is H or OH;
R20, R21, R26 and Rxe2x80x2 are as defined above with respect to Formula I;
p is 1, 2, 3 or 4; and
X is C, O, S or Nxe2x80x94Re where Re is COH, CO2Rxe2x80x2 or SO2Rxe2x80x2,
and the pharmaceutically acceptable salts thereof.
In a preferred aspect, the invention relates to the C26 des-methyl analogue of Formula IIa: 
and to pharmaceutical compositions and methods of treatment therewith.
Still another aspect of the invention relates to the C26 des-methyl homologues of the native bryostatins, as illustrated in Formula VI: 
where ORA and RB correspond to the naturally occurring bryostatin substituents, including:
such as C26 des-methyl Bryostatin 1, the compound of Formula VIa: 
the C26 des-methyl homologues of the native bryostatins, as illustrated in Formula VII: 
where RC and RD correspond to the naturally occurring bryostatin substituents, including:
and to the C26 des-methyl homologues of the native Bryostatin 3, as illustrated in the following formula: 
and to pharmaceutical compositions and methods of treatment therewith.
Excluded from the scope of the invention are the analogues of Formula 1998a (where R3 is H or OH) and the analogue of Formula 1998b: 
In another aspect, the invention relates to a pharmaceutical composition containing a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof admixed with at least one pharmaceutically acceptable excipient.
In still another aspect, the invention relates to a method of treating hyperproliferative cellular disorders, particularly cancer in a mammal by administering to a mammal in need of such treatment a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, either alone or in combination with a second agent, preferably a second anti-cancer agent that acts by a distinct mechanism vis-a-vis the mechanism of the compound of Formula I.
In yet another aspect, the invention relates to methods of treatment for a mammal having an immune-related disease or receiving immunosuppressive therapy, by administering of a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.
In another aspect of the invention, there is provided a method for the synthesis of bryostatin analogues, including the steps of esterification and macrotrasacetylization of a protected recognition domain with a protected linker synthon, followed by deprotection. Particularly preferred is reduction of a C26 OBn protected predursor to give the corresponding C26 des-methyl bryostatin analogue. A related aspect of the invention entails the novel products made by the foregoing process.
The invention also includes pharmaceutical compositions containing one or more compounds in accordance with the invention.
In another aspect, the invention includes a method of inhibiting growth, or proliferation, of a cancer cell. In the method, a cancer cell is contacted with a bryostatin analogue compound in accordance with the invention in an amount effective to inhibit growth or proliferation of the cell. In a broader aspect, the invention includes a method of treating cancer in a mammalian subject, especially humans. In the method, a bryostatin analogue compound in accordance with the present invention is administered to the subject in an amount effective to inhibit growth of the cancer in the patient.
These and other objects and features of the invention will be better understood in light of the following detailed description.
As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
As used herein, the terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynyl,xe2x80x9d refer to saturated and unsaturated monovalent moieties in accordance with their standard meanings, including straight-chain, branched-chain and cyclic moieties, optionally containing one or more intervening heteroatoms, such as oxygen, sulfur, and nitrogen in the chain or ring, respectively. Exemplary alkyl groups include methyl, ethyl, isopropyl, cyclopropyl, 2-butyl, cyclopentyl, and the like. Exemplary alkenyl groups include 2-pentenyl, 2,4-pentadienyl, 2-octenyl, 2,4,6-octatrienyl, CH3xe2x80x94CH2xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, cyclopentadienyl, and the like. Exemplary alkynyl groups include CH3Cxe2x89xa1CCH2xe2x80x94, 4-pentyn-1-yl, and the like. Exemplary cyclic moieties include cyclopentyl, cyclohexyl, furanyl, pyranyl, tetrahydrofuranyl, 1,3-dioxanyl, 1,4-dioxanyl, pyrrolidyl, piperidyl, morpholino, and reduced forms of furanyl, imidazyl, pyranyl, pyridyl, and the like.
xe2x80x9cLower alkylxe2x80x9d, xe2x80x9clower alkenylxe2x80x9d, and xe2x80x9clower alkynylxe2x80x9d refer to alkyl, alkenyl, and alkynyl groups containing 1 to 4 carbon atoms.
The term xe2x80x9carylxe2x80x9d denotes an aromatic ring or fused ring structure of carbon atoms with no heteroatoms in the ring(s). Examples are phenyl, naphthyl, anthracyl, and phenanthryl. Preferred examples are phenyl and napthyl.
The term xe2x80x9cheteroarylxe2x80x9d is used herein to denote an aromatic ring or fused ring structure of carbon atoms with one or more non-carbon atoms, such as oxygen, nitrogen, and sulfur, in the ring or in one or more of the rings in fused ring structures. Examples are furanyl, pyranyl, thienyl, imidazyl, pyrrolyl, pyridyl, pyrazolyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalyl, and quinazolinyl. Preferred examples are furanyl, imidazyl, pyranyl, pyrrolyl, and pyridyl.
xe2x80x9cAralkylxe2x80x9d and xe2x80x9cheteroaralkylxe2x80x9d refer to aryl and heteroaryl moieties, respectively, that are linked to a main structure by an intervening alkyl group, e.g., containing one or more methylene groups.
xe2x80x9cAlkoxyxe2x80x9d, xe2x80x9calkenoxyxe2x80x9d, and xe2x80x9calkynoxyxe2x80x9d refer to an alkyl, alkenyl, or alkynyl moiety, respectively, that is linked to a main structure by an intervening oxygen atom.
It will be appreciated that the alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl moieties utilized herein can be unsubstituted or substituted with one or more of the same or different substituents, which are typically selected from xe2x80x94X, xe2x80x94Rxe2x80x2, xe2x95x90O, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x95x90S, xe2x80x94NRxe2x80x2Rxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x2Rxe2x80x2+, xe2x95x90NRxe2x80x2, xe2x80x94CX3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NCO, xe2x80x94NCS, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94S(O)2Oxe2x88x92, xe2x80x94S(O)2OH, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(O)X, xe2x80x94C(S)Rxe2x80x2, xe2x80x94C(S)X, xe2x80x94C(O)ORxe2x80x2, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)ORxe2x80x2, xe2x80x94C(O)SRxe2x80x2, xe2x80x94C(S)SRxe2x80x2, xe2x80x94C(O)NRxe2x80x2Rxe2x80x2, xe2x80x94C(S)NRxe2x80x2Rxe2x80x2 and xe2x80x94C(NR)NRxe2x80x2Rxe2x80x2, where each X is independently a halogen (F, Cl, Br, or I, preferably F or Cl) and each Rxe2x80x2 is independently hydrogen, alkyl, alkenyl, or alkynyl. In one embodiment, Rxe2x80x2 is lower alkyl, lower alkenyl, or lower alkynyl. NRxe2x80x2Rxe2x80x2 also includes moieties wherein the two Rxe2x80x2 groups form a ring with the nitrogen atom.
While practical size limits for the various substituent groups will be apparent to those skilled in the art, generally preferred are the alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl moieties containing up to about 40 carbon atoms, more preferably up to about 20 carbon atoms and most preferably up to about 10 carbon atoms (except as otherwise specifically noted, for example, with reference to the embodiment of the invention where a preferred R20 substituent has about 7 to 20 carbon atoms).
As to any of the above groups that contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers and mixtures thereof arising from the substitution of these compounds.
Except as otherwise specifically provided or clear from the context, the term xe2x80x9ccompoundsxe2x80x9d of the invention should be construed as including the xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d thereof (which expression has been eliminated in certain instances for the sake of brevity).
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to salts which retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable. In some cases, the compounds of this invention are capable of forming acid and/or base salts, derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
xe2x80x9cMammalxe2x80x9d is intended to have its conventional meaning. Examples include humans, mice, rats, guinea pigs, horses, dogs, cats, sheep, cows, etc.
The term xe2x80x9ctreatmentxe2x80x9d or xe2x80x9ctreatingxe2x80x9d means any treatment of a disease in a mammal, including:
preventing the disease, that is, causing the clinical symptoms of the disease not to develop;
inhibiting the disease, that is, arresting the development of clinical symptoms; and/or
relieving the disease, that is, causing the regression of clinical symptoms.
The term xe2x80x9ceffective amountxe2x80x9d means a dosage sufficient to provide treatment for the disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.
The present invention provides new analogues of bryostatin that can be synthesized conveniently in high yields and which have useful biological activities. The compounds of the invention can be broadly described as having two main regions that are referred to herein as a xe2x80x9crecognition domainxe2x80x9d (or pharmacophoric region) and a relatively lipophilic xe2x80x9cspacer domainxe2x80x9d (or linker region). The recognition domain contains structural features that are analogous to those spanning C17 through C26 to C1, including the C ring formed in part by atoms C19 through C23, and the lactone linkage between C1 and C25 of the native bryostatin macrocycle. The spacer domain, on the other hand, joins the atoms corresponding to C1 through C17 of the native bryostatin macrocycle to substantially maintain the relative distance between the C1 and C17 atoms and the directionality of the C1C2 and C16C17 bonds, as illustrated by the arrows and distance xe2x80x9cdxe2x80x9d in Formula Ia (in which the substituent groups are as defined with reference to Formula I). 
In addition to its function of maintaining the recognition domain in an active conformation, the spacer domain (shown as xe2x80x9cLxe2x80x9d in Formula la and sometimes also referred to as a linker region) provides a moiety that can be readily derivatized according to known synthetic techniques to generate analogues having improved in vivo stability and pharmacological properties (e.g., by modulating side effect profiles) while retaining biological activity.
It has been found in the present invention that the linker region of of the bryostatin family can be varied significantly without eliminating activity. Thus, a wide variety of linkers can be used while retaining significant anticancer and PKC-binding activities. Preferably, the compounds of the present invention include a linker moiety L, which is a linear, cyclic, or polycyclic linker moiety containing a continuous chain of from 6 to 14 chain atoms, one embodiment of which defines the shortest path from C25 via C1 to C17. Thus, L may consist solely of a linear chain of atoms that links C17 via C1 to C25, or alteratively, may contain one or more ring structures which help link C17 via C1 to C25. Preferably, the linker region includes a lactone group (xe2x80x94C(xe2x95x90O)Oxe2x80x94), or a lactam group (xe2x80x94C(xe2x95x90O)NHxe2x80x94), which is linked to C25 of the recognition region, by analogy to the C1 lactone moiety that is present in the naturally occurring bryostatins. In addition, it is preferred that the linker include a hydroxyl group analogous to the C3 hydroxyl found in naturally occurring bryostatins, to permit formation of an intramolecular hydrogen bond between the C3 hydroxyl of the linker and the C19 hydroxyl group of the recognition region (and optionally with the oxygen of the native B ring). In one preferred embodiment, the linker terminates with xe2x80x94CH(OH)CH2C(xe2x95x90O)Oxe2x80x94, for joining to C25 of the recognition region via an ester (or when cyclized a lactone) linkage.
In one embodiment of the invention where R26 is H, the compounds of the invention differ from known bryostatins and bryostatin analogues in that the present compounds contain a primary alcohol moiety at C26, i.e., the present analogues lack a methyl group corresponding to the C27 methyl that is ordinarily present in naturally occurring bryostatins. Surprisingly, while the C27 methyl moiety was previously believed to limit rotation of the C26 alcohol and contribute to PKC binding affinity, it has been found that this structural modification can significantly increase PKC binding and also increases efficacy against cancer cells. Other modifications of R26 are provided to further modulate these characteristics, as are the C26 des-methyl homologues of the native bryostatins.
In another aspect, the present invention provides bryostatins and bryostatin analogues in which R20 is longer (e.g., having 9 to 20 or more carbon atoms) than the corresponding substituents at C20 in the native bryostatins (e.g., Bryostatin 3 having an 8-carbon atom moiety).
Certain preferred spacer domains employed in the compounds of the invention are illustrated in Formulae II through V: 
wherein:
R3 is H, OH or a protecting group;
R6 is H, H or xe2x95x90O;
R8 is selected from the group: H, OH, Rxe2x80x2, xe2x80x94(CH2)nO(O)CRxe2x80x2 or (CH2)nCO2-haloalkyl, where n is 0, 1, 2, 3, 4 or 5;
R9is H or OH;
R20, R21, R26 and Rxe2x80x2 are as defined above with respect to Formula I;
p is 1, 2, 3 or 4; and
X is C, O, S or Nxe2x80x94Re where Re is a group that stabilizes the nitrogen""s lone pair of electrons, such as COH, CO2Rxe2x80x2 or SO2Rxe2x80x2,
the pharmaceutically acceptable salts thereof, and the corresponding lactams.
Excluded from the scope of the present invention are the compounds of formula 1998a where R3 is H or OH and where R20 is xe2x80x94Oxe2x80x94C(O)xe2x80x94CH3 or xe2x80x94Oxe2x80x94C(O)xe2x80x94(CH2)6xe2x80x94CH3, and the compounds of formula 1998b where R is H or t-Bu: 
For simplicity of reference, the compounds of Formulae I-V are named and numbered herein as corresponding to the naturally occurring bryostatin macrocycle, described above with reference to Formula A. For example, the C26 des-methyl homologue of native bryostatin 1 (a compound of the present invention) has the structure illustrated in Formula VIa: 
By way of comparison, the analogues of the invention in which R26 is hydrogen, such as those of Formula IIa and Formula IVa: 
are also referred to as xe2x80x9cC26 des-methylxe2x80x9d, notwithstanding that the structures corresponding to L (in Formula I) or the corresponding spacer domain (in Formulae II-V), or even the recognition domain, contain fewer carbon atoms than native bryostatin such that the xe2x80x9cC26xe2x80x9d position would be assigned a lower number were these analogues to be named without reference to the native structure.
The terms xe2x80x9csolventxe2x80x9d, xe2x80x9cinert organic solventxe2x80x9d or xe2x80x9cinert solventxe2x80x9d mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (xe2x80x9cTHFxe2x80x9d), dimethylformamide (xe2x80x9cDMFxe2x80x9d), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like]. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.
The terms xe2x80x9cprotecting groupxe2x80x9d or xe2x80x9cblocking groupxe2x80x9d refer to any group which when bound to a functional group such as one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group. The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
The term xe2x80x9cq.s.xe2x80x9d means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to the desired volume (i.e., 100%).
Unless specified to the contrary, the reactions described herein take place at atmospheric pressure within a temperature range from about 5xc2x0 C. to 100xc2x0 C. (preferably from 10xc2x0 C. to 50xc2x0 C.; most preferably at xe2x80x9croomxe2x80x9d or xe2x80x9cambientxe2x80x9d temperature, e.g., 25xc2x0 C.). Further, unless otherwise specified, the reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about 5xc2x0 C. to about 100xc2x0 C. (preferably from about 10xc2x0 C. to about 50xc2x0 C.; most preferably about 25xc2x0 C.) over a period of about 0.5 to about 10 hours (preferably about 1 hour). Parameters given in the Examples are intended to be specific, not approximate.
Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, distillation, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the general description and examples. However, other equivalent separation or isolation procedures can, of course, also be used.
The compounds of the invention may be produced by any methods available in the art, inducing chemical and biological (e.g., recombinant and in vitro enzyme-catalyzed) methods. In one embodiment, the present invention provides a convergent synthesis in which subunits primarily corresponding to the recognition and spacer domains are separately prepared and then joined by esterification-macrotransacetalization (Wender et al., 1998a, 1998b, 1998c). Additional syntheses of the compounds of Formulae I-VI are described below with reference to the Reaction Schemes.
Reaction Scheme 1 illustrates synthesis of precursors for the recognition domain in compounds of the invention. 
Reaction Scheme 2 illustrates the further synthesis of recognition domains for C26 des-methyl compounds of the invention. 
Reaction Scheme 3 illustrates synthesis of the protected alcohol precursor to many of the C26 methyl analogues of the invention. 
Reaction Scheme 4 illustrates the synthesis of linker synthons for preparing the compounds of Formula II. 
Reaction Scheme 5A illustrates the synthesis of linker synthons for preparing the compounds of Formula III where R9 is OH. 
Reaction Scheme 5B illustrates the synthesis of linker synthons for preparing the compounds of Formula III where R8 and/or R9 are H. 
Reaction Scheme 5C illustrates the synthesis of linker synthons for preparing the compounds of Formula III where R6 is xe2x95x90O with a variety of possible substituents at R8. 
Reaction Scheme 6 illustrates the synthesis of linker synthons for preparing the compounds of Formula IV. 
Reaction Scheme 7A illustrates the synthesis of the compounds of Formula II. 
Reaction Scheme 7B illustrates the synthesis of the compounds of Formula III. 
Reaction Scheme 8 illustrates synthesis of the Compounds of Formula IV, particularly where R26 is methyl, the C26 des-methyl analogues of Formula IV being obtained by like synthesis. 
Reaction Scheme 9 illustrates synthesis of the Compounds of Formula V. 
Reaction Schemes 10 and 11 illustrate the synthesis of compounds of the invention that are further derivatized at the C20 position, as discussed in Examples 4B, 4C and 4D. 
Starting Materials. Conveniently, compounds of the invention can be prepared from starting materials that are commercially available or may be readily prepared by those skilled in the art using commonly employed synthetic methodology.
Reaction Scheme 1 illustrates a method for forming a synthon designated herein as 111 which is useful for providing the recognition domain in compounds of the invention, for example as detailed in Example 1. 6-(Tert-butyldimethylsilylhydroxy)-5-dimethylhexane-2,4-dione (101, Example 1B) is stirred with 2 equivalents of LDA (lithium diisopropylamine) in THF (tetrahydrofuran), followed by addition of 0.9 equivalents of 3R-p-methoxybenzyl-4R-benzylhydroxypentane-1-a1 (102, Example 1A) to afford diasteriomeric aldol mixture 103 after suitable purification. To 103 is then added a catalytic amount of p-methylphenylsulfonic acid (p-TsOH) with stirring at room temperature followed by base quenching to produce pyranone condensation product 104 as a mixture of xcex1 and xcex2-isomers at C23 (104a and 104b). The xcex2-isomer (104a) is separated from the xcex1-isomer and is reacted with NaBH4 in the presence of CeCl3.7H2O, followed by quenching with aqueous brine to form an allylic alcohol (not shown)that can then be epoxidized with m-chloroperbenzoic acid (mCPBA) in 2:1 CH2Cl2:MeOH containing sodium bicarbonate as a buffer to yield a C19-methoxylated C20,C21 syn-diol 105. Selective benzoylation of the C21 equatorial alcohol with benzoyl chloride to afford C21 monobenzoate (not shown), followed by oxidation of the C20 hydroxyl with Dess-Martin periodinane (1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one) at room temperature affords the corresponding 20-keto-21-benzoate product 106. Treatment of 106 with SMI2 (2 equiv) yields a ketone 107 selectively deoxygenated at C21. Next, ketone 107 is reacted with LDA and OHCCO2CH3 in THF at xe2x88x9278xc2x0 C. to afford aldol mixture 108. After purification, 108 is reacted with methanesulfonylchloride in CH2Cl2 containing triethylamine, followed by reaction with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in THF to effect an aldol condensation and elimination of water, to yield an xcex1,xcex2-unsaturated methyl ester (enone 109) with an E-stereoconfiguration. Treatment of enone 109 with NaBH4 in the presence of CeCl3.7H2O produces exclusively the C20 axial alcohol 110. This product can then be esterified at C20, with octanoic acid for example, to yield the desired synthon 111.
It will be appreciated how the foregoing procedures can be exploited or modified to produce recognition region synthons having different substituents. For example, compounds where R21 contains a C35 ester group having a Z-configuration are produced during formation of intermediate 109 (Example 1C) and can be isolated by chromatography. Similarly, other ester groups can be introduced at C35 by replacing the OHCCO2CH3 reactant used to form 108 above with an appropriately substituted compound of the form OHCCO2Rxe2x80x2, in which Rxe2x80x2 is other than methyl.
In addition, as detailed below, other substituents can be introduced in synthon 111 to generate substituent R20 at C20 by substituting any of a variety of carboxylic acids for the octanoic acid reacted with axial alcohol 110 (as in the last step of Example 1C), including other saturated, unsaturated, aryl, and carboxylic acids. In synthesizing the compounds of the invention where R20 has been varied, the substituent (e.g., a desired C20 ester substituent) can be introduced into a recognition region synthon prior to condensing the recognition region synthon with a linker synthon using the procedures described in Example 4. In Example 4A, the C20 octanoate substituent in synthon 111 can be replaced with an acetyl group by first protecting the base labile aldehyde group using trimethyl orthorformate to form the dimethyl acetal. The C20 octanoate ester can then be cleaved using a basic solution, such as K2CO3 in methanol, to afford the free C20 alcohol, followed by reaction with an activated form of acetic acid, such as acetic anhydride or acetyl chloride, to obtain the C20 acetate product. The product can then be deprotected at the C15 aldehyde, C19 oxygen, and C25 oxygen using benzoquinone compound DDQ (to remove the p-methoxybenzyl group and cleave the dimethyl acetal) followed by aqueous HF (demethylation at C19) to afford the corresponding C19 alcohol. This product can then be condensed with an appropriately substituted linker synthon to produce a desired bryostatin analogue, such as analogue 702.1, as detailed in Example 4A.
The protected alcohol precursor to many of the C26-desmethyl bryostatin analogues of the invention (the compounds of Formula I where R26 is H) can be made as illustrated in Reaction Scheme 2. Di(benzyl ether) 111 can be hydrogenated over Pearlman""s catalyst to produce the corresponding C25,C26 diol 201. Treatment of the diol with lead tetraacetate yields the corresponding C25 aldehyde (not shown), with the release of C26 and C27. Reaction of the aldehyde with Cp2Ti(Cl)CH2Al(CH3)2 (Tebbe""s reagent) yields C25,C26 olefin 202. Alternatively, sodium periodate can be used in place of lead tetraacetate.
Treatment of olefin 202 with HF/pyridine is effective to remove the silyl protecting group, followed by treatment with Dess-Martin periodinane (supra) to oxidize the C17 alcohol to an aldehyde group, affording aldehyde 203. The C25,C26 olefin of 203 can be converted to C25,C26 diol 204 by reaction with chiral dihydroxylating reagent (DHQD)2AQN in the presence of K3Fe(CN)6, K2CO3 and K2OsO2(OH)4 in t-butanol. Product 204 is obtained as a 2:1 (xcex1:xcex2) mixture of 25-hydroxy diastereomers. The xcex1-diastereomer can be removed later in the synthesis. Treatment of 204 with triethylsilyl chloride yields protected diol 205, which can be employed in the synthesis of the compounds of Formula V.
Addition of backbone atoms corresponding to C15 and C16 of the bryostatin backbone to 205 can be accomplished in four steps. First, the C17 aldehyde is allylated with allyl diethylborane. The reaction is quenched with saturated sodium bicarbonate to yield the desired C17 allyl adduct. The C17 hydroxyl group can then be acylated with acetic anhydride in the presence of triethylamine and 4-dimethylaminopyridine (DMAP), to afford a diastereomeric mixture of homoallylic C17 acetates. This product mixture can be oxidized using N-methylmorpholine N-oxide and osmium tetraoxide, followed by neutralization with sodium bicarbonate. After extraction, the residue is reacted with lead tetraacetate, followed by addition of DBU to cause elimination of the acetate group, yielding enal 206.
The C25 hydroxyl group of 206 can be unmasked in preparation for closure of the macrocycle as follows. First, enal 206 is treated with aqueous hydrofluoric acid to provide a crude diol intermediate in which the C19 methoxy group is converted to a free hydroxyl. Next, the diol product is reacted with tert-butyldimethylsilyl chloride (TBSCl) in the presence of imidazole to produce alcohol 207 containing a C25 hydroxyl group and C26 OTBS group as a 2:1 (xcex2:xcex1) mixture of C25 diastereomers. Silica gel chromatography can be used to resolve the diastereomers, affording the xcex2 diastereomer in 50-60% yield.
The protected alcohol precursor to many of the C26-methyl bryostatin analogues of the invention (Formula I where R26 is methyl) can be made as illustrated in Reaction Scheme 3, via methods analogous to the preparations for 205 and 206. Deprotection and acylation of formula 111 may be accomplished, for example, by following the procedures described in Wender et al., (1998a).
Linker synthons for the compounds of Formula II (where X is a heteroatom) can be prepared, for example, as illustrated with reference to Reaction Scheme 4, and later described in Examples 2A and 2B. These compounds contain two rings that are analogous to the A and B rings of bryostatin, but lack the naturally occurring substituents at C7, C8, C9, and C13. The presence of a heteroatom, such as an oxygen, sulfur or nitrogen atom (the lone electron pair of which is stabilized) in place of C14 does not adversely affect activity of the end product, but is required for transacetylization in the later synthetic steps. The compounds of formulae 406 and 408 differ in that 402 provides a protecting group precursor for a hydroxyl group attached to C3, whereas 406 does not provide for a hydroxyl at C3.
The linker synthons for the compounds of Formula III (in which X is a heteroatom), which contain a B-ring-like structure but lack an A-ring, are prepared, for example, as illustrated with reference to Reaction Schemes 5A through 5C. Examples 2C and 2D describe methods for preparing synthons 504 and 508. In both examples, R8 is a tert butyl group attached to C9. However, with reference to the preparation of 505, the t-BuLi reactant can be replaced by Rxe2x80x2Li to generate the corresponding linker synthons of 508 where R8 is Rxe2x80x2. 504 additionally contains a TMS protecting group for synthesis of the compounds where R9 is a hydroxyl attached to C9, rather than hydrogen. Also, both synthons contain a TBS protecting group for the compounds where R3 is a hydroxyl group attached to C3. Example 2E describes the corresponding method for making synthon 507, which is unsubstituted at C9. Example 2G describes a method for preparing linker synthons in which C5 is provided as an ester carbonyl. In addition, the synthons in this Example contain an R6 substituent that is preferably a saturated or unsaturated substituent containing 1 to 20 carbon atoms and optionally (1) one or more oxygen atoms and (2) optionally one or more nitrogen atoms. In synthon 514 in Example 2G, R8 is xe2x80x94C(CH3)2CH2OC(xe2x95x90O)C13H27. However, other R6 substituents can be introduced by suitable modification of the procedure as will be evident to one of ordinary skill in the chemical arts.
Synthesis of a completely acyclic linker synthon 606 (where neither an A- nor a B-ring-like structure is present) is described with reference to Reaction Scheme 6 and in Example 2F.
As illustrated with reference to Reaction Scheme 7A, and further described in Example 3A, an alcohol such as 207, 303 or 304 is reacted with an acid such as 406 or 408 in a two step process to form the desired macrocyclic structure. After in situ conversion of the acid (408) to a mixed anhydride, the alcohol (207) is added to form ester 701. The ketal portion of 408 is then joined (in a process referred to as macrotranacetylization) to C15 of 701 by adding 70% HF/pyridine hydrofluoric acid to catalyze cleavage of the menthone ketal, cleavage of the TBS ethers at C3 and C26, and formation of a new ketal between the C15 aldehyde group and the linker diol moiety generated by release of the menthone (where X is oxygen), to afford desired analogue of Formula II where X is a heteroatom and R26 is H (starting with alcohol 207) or methyl (starting with 303 or 304), i.e., compound of formula 702. This last reaction is also effective to set the stereocenter at C15 to a thermodynamically preferred configuration. The analogous synthesis of compounds of Formula III (where X is a heteroatom), first forming the ether bond between C1 and C25, is illustrated with reference to Reaction Scheme 7B (where formula 703 corresponds to any of formulae 504, 507, 508 or 513) and further described in Example 3C.
As illustrated with reference to Reaction Scheme 8, and further described in Example 3B, the compounds of Formula IV (such as formula 807) can be made from pharmacophoric synthon 801 and linker synthon 606 from Example 2F.
Reaction Scheme 9 illustrates synthesis of the compounds of Formula V, e.g., as further described in Example 3D, from synthon 111 and an activated dicarboxylic acid (succinic anhydride) to give formula 903.
Although the bryostatin analogues produced in Examples 3B, 3C and 3D all contain a C27 methyl group, analogous C26 desmethyl analogues can be readily synthesized using an appropriate C26 desmethyl synthon, such as C26 desmethyl synthon 207 described in Example 1C. Compounds of the invention having a naturally occurring bryostatin backbone (e.g., including a naturally occuring linker region), but lacking the C27 methyl group, can also be prepared by adapting synthetic protocols published by Kageyama et al. (1990) and Evans et al. (1998), which are incorporated herein by reference. In brief, the published methods are modified by utilizing synthons in which the C27 methyl group has been omitted, to afford the desired C26-desmethyl analogues.
As illustrated with reference to Reaction Scheme 10, synthesis of a C20 heptanoate ester 43 is described in Example 4B, using a similar reaction scheme to that employed in Example 4A, except that heptenoic acid in the presence of triethylamine, DMAP, and Yamaguchi""s agent is used in place of acetic anhydride. Yamaguchi""s reagent is again employed in step f to activate the COOH group of formula 6, followed by removal of the TBS group in step g, hydrolysis of the menthone and transacetylization in step h, and saturation of the double bond upon removal of the benzyl group by hydrogenolysis in step i. Synthesis of a C20 myristate ester analogue 48 (14 carbon atom chain) is illustrated with reference to Reaction Scheme 11 and described in Example 4C. Reaction Scheme 11 and Example 4D describes synthesis of a bryostatin analogue containing an aryl ester group (benzoate) at C20, by suitable adaptation of the procedure in Example 1C for making compound 207. It will be appreciated how these procedures can be modified to introduce other C20 esters by substituting the starting materials necessary to produce the desired products. In particular, C26 des-methyl analogues can be made using an appropriate C26 des-methyl synthon, such as 207 noted above.
The lactam analogues of the invention are obtained by converting the C25 hydroxyl group (e.g., of formula 207) to an amine under Mitsonobu conditions, after first protecting the aldehyde (and the C19 hydroxyl group in the corresponding compounds in Reaction Schemes 10 and 11) followed by formation of the macrocycle and de-protection under conditions analogous to those employed for the lactone analogues, as will be apparent to one skilled in the art.
The C26 des-methyl bryostatin homologues of the invention can be obtained by substituting homologous des-methyl starting materials for the starting materials employed in published bryostatin syntheses (e.g., Masamune 1988a, 1988b, Evans et al. 1998, Kageyama et al. 1990). For example, in the total synthesis of bryostatin 7, Serine is substituted for threonine in a Masamune""s C17-C26 southern bryostatin synthesis to yield the corresponding C26 des-methyl sulfone. Other synthetic methodology will be apparent to those skilled in the art given the objective of providing such C26 des-methyl bryostatin homologues.
A C19,C26 hydroxyl-protected, C26 des-methyl bryostatin recognition domaine precursor and an optionally protected linker synthon are esterified, macrotransacetylated and de-protected to give the corresponding C26 des-methyl bryostatin analogue.
A bryostatin analogue precursor having the C26 hydroxyl substituted by a protecting group (particularly OBn) is reduced to give the corresponding compound of Formula I.
Serine is substituted for threonine in a Masamune""s C17-C26 southern bryostatin synthesis to yield the corresponding C26 des-methyl sulfone, which in turn is employed in synthesis of a C26 des-methyl bryostatin homologue.
A compound of Formula I-VI is contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salt.
A pharmaceutically acceptable acid addition salt of Formula I-VI is contacted with a base to form the corresponding compound of Formula I-VI.
The following substituents, compounds and groups of compounds are presently preferred.
In the compounds of Formulae I-V, especially those of Formulae II-V, it is preferred that R26 is H. Most preferred are the compounds of Formula II where R26 is H, and of those where X is oxygen. Of the compounds where R26 is H, additionally preferred are those compounds where R20 is O2CRxe2x80x2, especially where Rxe2x80x2 is alkyl (preferably about C7-C20 alkyl), alkenyl (preferably about C7-C20 alkenyl such as CH3xe2x80x94CH2xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94) or aryl (preferably phenyl or naphthyl). Another group of preferred compounds where R26 is H are those where R21 is xe2x95x90CRaRb (especially where one of Ra or Rb is H and the other is CO2Rxe2x80x2, and preferably where Rxe2x80x2is C1-C10 alkyl, most preferably lower alkyl such as methyl).
The compounds of Formulae I-V, especially II-V, are preferred where R20 is O2CRxe2x80x2 and Rxe2x80x2 is alkyl (preferably about C7-C20 alkyl), alkenyl (preferably about C7-C20 alkenyl such as CH3xe2x80x94CH2xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94) or aryl (preferably phenyl or naphthyl). Particularly preferred are those compounds where R20 is O2CRxe2x80x2 and R21 is xe2x95x90CRaRb (especially where one of Ra or Rb is H and the other is CO2Rxe2x80x2, and preferably where Rxe2x80x2 is C1-C10 alkyl, most preferably lower alkyl such as methyl).
The compounds of Formulae I-V, especially II-V, are preferred where R21 is xe2x95x90CRaRb (especially where one of Ra or Rb is H and the other is CO2Rxe2x80x2, and preferably where Rxe2x80x2 is C1-C10 alkyl, most preferably lower alkyl such as methyl).
Of the compounds according to Formula I, it is preferred that L be a group having from about 6 to about 14 carbon atoms.
Of the compounds according to Formulae II or III, it is preferred that X is oxygen.
Of the compounds according to Formulae II-IV, it is preferred that R3 is OH, and especially preferred that X is oxygen in the case of Formulae II and III.
Of the compounds according to Formula II, it is preferred that R20 is O2CRxe2x80x2 where Rxe2x80x2 is alkyl (preferably about C7-C20 alkyl), alkenyl (preferably about C7-C20 alkenyl such as CH3xe2x80x94CH2xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94) or aryl (preferably phenyl or naphthyl). Of these, further preferred are the compounds where R21 is xe2x95x90CRaRb (especially where one of Ra or Rb is H and the other is CO2Rxe2x80x2, and preferably where Rxe2x80x2 is C1-C10 alkyl, most preferably lower alkyl such as methyl).
Of the compounds according to Formula III, it is preferred that R9 is H. It is further preferred that R8 is xe2x80x94(CH2)nO(O)CRxe2x80x2, particularly where Rxe2x80x2 is alkyl, R6 optionally being xe2x95x90O.
Further preferred are those compounds that combine various of the above-mentioned features. The single isomers highlighted in the reaction schemes and examples are also preferred.
Presently, most preferred is the compound of Formula II where X is oxygen, R3 is OH, R20 is xe2x80x94Oxe2x80x94COxe2x80x94C7H15, R21 is xe2x95x90CHxe2x80x94CO2Me and R26 is H.
The compounds of the present invention are useful as bryostatin-like therapeutic agents, and in pharmaceutical formulations and methods of treatment employing the same. Other compounds of the invention are useful a precursors in the synthesis of such agents. Importantly, in many cases, the compounds of the present invention can be readily synthesized on a large scale, and thus can be made readily available for commercial purposes as compared to the low yields and environmental problems inherrent in the isolation of bryostatins from natural sources.
In one aspect, the compounds of the invention find use as anticancer agents in mammalian subjects. For example, representative cancer conditions and cell types against which the compounds of the invention may be useful include melanoma, myeloma, chronic lymphocytic leukemia (CLL), AIDS-related lymphoma, non-Hodgkin""s lymphoma, colorectal cancer, renal cancer, prostate cancer, cancers of the head, neck, stomach, esophagus, anus, or cervix, ovarian cancer, breast cancer, peritoneal cancer, and non-small cell lung cancer. The compounds appear to operate by a mechanism distinct from the mechanisms of other anticancer compounds, and thus can be used synergistically in combination with other anticancer drugs and therapies to treat cancers via a multimechanistic approach. The compounds of the invention exhibit potencies comparable to or better than previous bryostatins against many human cancer types.
In another aspect, the compounds of the invention can be used to strengthen the immune system of a mammalian subject, wherein a compound of the invention is administered to the subject in an amount effective to increase one or more components of the immune system. For example, strengthening of the immune system can be evidenced by increased levels of T cells, antibody-producing cells, tumor necrosis factors, interleukins, interferons, and the like. Effective dosages may be comparable to those for anticancer uses, and can be optimized with the aid of various immune response assay protocols such as are known in the art (e.g., see Kraft, 1996; Lind, 1993; U.S. Pat. No. 5,358,711, all incorporated herein by reference). The compound can be administered prophyllactically, e.g., for subjects who are about to undergo anticancer therapies, as well as therapeutically, e.g., for subjects suffering from microbial infection, bum victims, subjects with diabetes, anemia, radiation treatment, or anticancer chemotherapy. The immunostimulatory activity of the compounds of the present invention is unusual among anticancer compounds and provides a dual benefit for anticancer applications. First, the immunostimulatory activity allows the compounds of the invention to be used in greater doses and for longer periods of time than would be possible for compounds of similar anticancer activity but lacking immunostimulatory activity. Second, the compounds of the present invention can offset the immunosuppressive effects of other drugs or treatment regimens when used in combination therapies. Additional features of the invention can be further understood from the following illustrative examples which are not intended to limit the scope of the invention in any way.
In practicing various aspects of the present invention, compounds in accordance with the invention can be tested for a biological activity of interest using any assay protocol that is predictive of activity in vivo. For example, a variety of convenient assay protocols are available that are generally predictive of anticancer activity in vivo.
In one approach, anticancer activity of compounds of the invention can be assessed using the protein kinase C assay detailed in Example 5. In this assay, Ki values are determined for analogues based on competition with radiolabeled phorbol 12,13-dibutyrate for binding to a mixture of PKC isoenzymes. PKC enzymes are implicated in a variety of cellular responses which may be involved in the activity of the bryostatins.
Example 6 describes another protein kinase C assay which can be used to assess the binding affinities of compounds of the invention for binding to the C1B domain of PKCxcex4. Although all PKC isozymes are upregulated immediately after administration of bryostatin or tumor promoting phorbol esters followed by an extended down-regulation period, PKCxcex4 appears to be protected against down regulation by bryostatin 1. Overexpression of PKCxcex4 inhibits tumor cell growth and induces cellular apoptosis, whereas depleting cells of PKCxcex4 can cause tumor promotion. Accordingly, this assay provides useful binding data for assessing potential anticancer activity.
Another useful method for assessing anticancer activities of compounds of the invention involves the multiple-human cancer cell line screening assays run by the National Cancer Institute (e.g., Boyd, 1989). This screening panel, which involves approximately 60 different human cancer cell lines, is a useful indicator of in vivo antitumor activity for a broad variety of tumor types (Grever et al., 1992, Monks et al., 1991), such as leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancers. Antitumor activites can be expressed in terms of ED50 (or GI50), where ED50 is the molar concentration of compound effective to reduce cell growth by 50%. Compounds with lower ED50 values tend to have greater anticancer activities than compounds with higher ED50 values. Example 7 describes a P388 murine lymphocytic leukemia cell assay which measures the ability of compounds of the invention to inhibit cellular growth.
Upon the confirmation of a compounds potential activity in the above in vitro assays, further evaluation is typically conducted in vivo in laboratory animals, for example, measuring reduction of lung nodule metastases in mice with B16 melanoma (e.g., Schuchter et al, 1991). The efficacy of drug combination chemotherapy can be evaluated, for example, using the human B-CLL xenograft model in mice (e.g., Mohammad et al, 1996). Ultimately, the safety and efficacy of compounds of the invention are evaluated in human clinical trials.
Experiments conducted in support of the present invention demonstrate that compounds of the present invention exhibit high potencies in several anticancer assays, as summarized in the Examples.
The invention includes a method of inhibiting growth, or proliferation, of a cancer cell, or enhancing the effectiveness of other drugs. In the method, a cancer cell is contacted with a bryostatin analogue compound in accordance with the invention in an amount effective to inhibit growth or proliferation of the cell. In a broader aspect, the invention includes a method of treating cancer in a mammalian subject, especially humans. In the method, a bryostatin analogue compound in accordance with the invention is administered to the subject in an amount effective to inhibit growth of the cancer in the patient. Similarly, in the immune modulation methods of the invention, a compound of the invention is administered to a subject in need thereof, in an amount herapeutically effective for bolstering of the immune system predisposed toward apoptosis.
Compositions and methods of the present invention have particular utility in the area of human and veterinary therapeutics. Generally, administered dosages will be effective to deliver picomolar to micromolar concentrations of the therapeutic composition to the target site. Typically, nanomolar to micromolar concentration at the target site should be adequate for many applications. Appropriate dosages and concentrations will depend on factors such as the particular compound or compounds being administered, the site of intended delivery, and the route of administration, all of which can be derived empirically according to methods well known in the art.
Administration of compounds of the invention in an appropriate pharmaceutical form can be carried out by any appropriate mode of administration. Thus, administration can be, for example, intravenous, topical, subcutaneous, transocular transcutaneous, intramuscular, oral, intra-joint, parenteral, peritoneal, intranasal, or by inhalation. The formulations may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, aerosols, and the like. In one embodiment, the formulation has a unit dosage form suitable for administration of a precise dose.
Pharmaceutical compositions of the invention typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, antioxidants, and the like. In one embodiment, the composition may comprise from about 1% to about 75% by weight of one or more compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, for example. Appropriate excipients can be tailored to the particular composition and route of administration by methods well known in the art, e.g., (Gennaro, 1990). Additional guidance for formulations and methods of administration can be found in patent references concerning previously known bryostatins, such as U.S. Pat. Nos. 4,560,774 and 4,611,066 to Pettit et al., which are incorporated herein by reference.
Usually, for oral administration, the compositions will take the form of a pill, tablet or capsule. Thus the composition will contain, along with active drug, a diluent such as lactose, sucrose, dicalcium phosphate, and/or other material, a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and/or derivatives thereof.
The compounds of the invention may also be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).
Liquid compositions can be prepared by dissolving or dispersing compound (e.g., from about 0.5% to about 20% of final volume), and optional pharmaceutical adjuvants in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution or suspension. Useful vehicles also include polyoxyethylene sorbitan fatty acid monoesters, such as TWEEN(trademark) 80, and polyethoxylated castor oils, such as Cremophor EL(trademark) available from BASF (Wyandotte, Md.), as discussed in PCT Publ. No. WO 97/23208 (which is incorporated herein by reference), which can be diluted into conventional saline solutions for intravenous administration. Such liquid compositions are useful for intravenous administration. One such formulation is PET diluent which is a 60/30/10 v/v/v mixture of PEG 400, dehydrated ethanol, and TWEEN(trademark)-80. Liquid compositions may also be formulated as retention enemas.
The compounds of the invention may also be formulated as liposomes using liposome preparation methods known in the art. Preferably, the liposomes are formulated either as small unilamellar vesicles or as larger vesicles.
If desired, the composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and antioxidants.
For topical administration, the composition is administered in any suitable format, such as a lotion or a transdermal patch. For delivery by inhalation, the composition can be delivered as a dry powder (e.g., Inhale Therapeutics) or in liquid form via a nebulizer.
Methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Gennaro (1990). The composition to be administered will, in any event, contain a quantity of one or more compounds of the invention in a pharmaceutically effective amount for relief of the condition being treated.
The compounds of the invention may also be introduced in a controlled-release form, for long-term delivery of drug to a selected site over a period of several days or weeks. In this case, the compound of the invention is incorporated into an implantation device or matrix for delayed or controlled release from the device.
The compounds can be incorporated in a biodegradable material, such as a biodegradable molded article or sponge. Exemplary biodegradable materials include matrices of collagen, polylactic acid-polyglycolic acid, and the like. In preparing bryostatin compounds in matrix form, the compounds may be mixed with matrix precursor, which is then crosslinked by covalent or non-covalent means to form the desired matrix. Alternatively, the compound can be diffused into a preformed matrix. Examples of suitable materials for use as polymeric delivery systems have been described e.g., Aprahamian, 1986; Emmanuel, 1987; Friendenstein, 1982; and Uchida, 1987.
Generally, compounds of the invention are administered in a therapeutically effective amount, i.e., a dosage sufficient to effect treatment, which may vary depending on the individual and condition being treated. Typically, a therapeutically effective daily dose is from 0.1 xcexcg/kg to 100 mg/kg of body weight per day of drug. Given the high therapeutic activities of the compounds of the invention, daily dosages of from about 1 xcexcg/kg and about 1 mg/kg of body weight may be adequate, although dosages greater than or less than this range can also be used.
It will be appreciated that the compounds of the invention may be administered in combination (i.e., together in the same formulation or in separate formulations administered by the same or different routes) with any other anti-cancer regimen deemed appropriate for the patient. For example, the compounds of the invention may be used in combination with other anticancer drugs such as vincristine, cisplatin, ara-C, taxanes, edatrexate, L-buthionine sulfoxide, tiazofurin, gallium nitrate, doxorubicin, etoposide, podophyllotoxins, cyclophosphamide, camptothecins, dolastatin, and auristatin-PE, for example, and may also be used in combination with radiation therapy. In a preferred embodiment, the combination therapy entails co-administration of an agent selected from: ara-C, taxol, cisplatin and vincristine.